1. Field of the Invention
The present invention concerns a flow type measuring apparatus which has enzyme electrodes, and measures concentrations of two different substances. More specifically, the invention relates to an improvement of the apparatus, having two enzyme electrodes which ultimately detect the same material, and on one enzyme electrode thereof, a first substance to be measured is yielded from a second substance by enzymatic reaction.
2. Description of the Prior Art
In recent years, immobilized enzymes have been broadly applied to the fields of clinical testing, fermenation and analytical chemistry. Especially, immobilized enzyme electrodes are frequently utilized, because of their high selectivity, readiness and easiness of measuring.
Recently, there have been efforts to develop the apparatus measuring two different substances. For instance, apparatus were introduced, which measures simultaneously;
(1) glucose and uric acid (the Japanese Laid-open Patent Publication No. 24142/1987),
(2) lactic acid and Pyruvic acid (the Japanese Laid-open Patent Publication No. 5172/1987),
(3) glucose and latic acid, or glucose and choline (GB 2063479), and so on. According to these prior arts, however, each enzyme immobilized on the electrode catalyzes the different reaction respectively.
On the contrary, in the case of common reaction, which yields or consumes the electrode active material (for example, ultimately hydrogen peroxide is formed, and the amount of the hydrogen peroxide is measured), the concentration of one substance (e.g. glucose) can be measured by one electrode, but the other electrode detects not only hydrogen peroxide originated from the other substance (e.g. sucrose) but also hydrogen peroxide originated from one substance (glucose). So one substance (glucose) can be measured, but the result of measurement of the other is addition of the values of two substances.
For example, in the case of measuring sucrose concentration in the sample containing both glucose and sucrose using the enzyme electrodes, the result is effected by glucose concentration. ##STR1##
Generally in order to measure sucrose concentration, sucrose is hydrolyzed to fructose and .alpha.- D-glucose by invertase (formula (i)). Thereafter .alpha.- D - glucose is converted to .beta.- D - glucose by mutarotase (formula (ii )). And hydrogen peroxide, that is electrode active material, is formed as a result of oxidation of .beta.- D- glucose catalyzed by glucose oxidase. This hydrogen peroxide is detected by the enzyme electrode electrochemically (C. Bertrand P. R. Coulet, D. C. Gautheron, Anal. Chim, Acta, 126,23-34(1981). But according to this method, the enzyme electrode measures not only .beta.- D- glucose converted from sucrose by formula (i) and (ii), but also .beta.- D - glucose originally contained in the sample, because .beta.- D - glucose originally contained in the sample yields hydrogen peroxide by the formula (iii), as well as .beta.- D - glucose formed by the formula (i), (ii ) does.
Therefore, in order to measure correct concentration of only sucrose, it is necessary;
to provide the layer containing glucose oxidase and catalase, in which glucose is oxidized by the glucose oxidase and hydrogen peroxide is removed by the catalase enzymatically before measuring sucrose(F. Scheller, R. R enneberg, Anal. Chim. Acta, 152, 265-269(1983)), or
to employ enzymatic reaction and electrochemical techinique in order to remove influence of glucose originally contained in the sample (the Japanese Laid-open Patent Publication No. 216947/1983).
But the apparatus employed in the above described method have some drawbacks that it is extremely difficult to immobilize the enzymes to the membrane in preparing the enzyme electrode and that the electrode itself must be so complicated. Furthermore, in order to measure two different substances, that is glucose as well as sucrose, another electrode must be provided and glucose concentration is measured by this electrode.
Recently reported was a flow type measuring apparatus, which comprises a column reactor cycling a sample solution (M. Massoom, A. Townshend, Anal. Chim. Acta, 171, 185-194(1985)). This apparatus, however, is complicated and far greater amount of enzyme is needed to compare with the apparatus having the enzyme electrodes. And it also had a drawback in that it gives a result incorrectly because of choking of the column.
The case of measuring the sample containing both sucrose and glucose is described above, however, when the same electrode active material is formed by the common enzymatic reaction on the two enzyme electrodes, in measuring two different substances simultaneously, for example in measuring maltose and glucose, or ester type cholesterol and free cholesterol, the same kinds of problems are caused.
So the inventors employed a measurement of concentrations of two different substances, for example glucose as the first substance to be measured, and sucrose as the second substance to be measured, using two enzyme electrodes by following method. A flow type measuring apparatus is prepared, which is provided with a first enzyme electrode having immobilized glucose oxidase for measuring glucose, and a second enzyme electrode having immobilized glucose oxidase, mutarotase and invertase for measuring sucrose.
A detected glucose concentration by the first enzyme electrode is calculated, and correct concentration of sucrose only is given by subtracting the result of the calculation from the output current value of the second enzyme electrode.
According to this method, however, when glucose concentration of the sample is comparatively high, because of converting sucrose to glucose on the second enzyme electrode, a great amount of the electrode active material is formed. Therefore, the amount exceeds the range, in which the amount of the electrode active material is proportional to the output current. Thus, an accurate value of sucrose concentration can not be obtained.
In this case, by using the second enzyme electrode having reduced amount of immobilized enzymes and lower sensitivity, the sample of high substance concentration is able to be analyzed. On the other hand, in this method, one must prepare the electrodes of different sensitivity corresponding to concentration of substances in the sample solution. So the apparatus employing this method is not practical at all.